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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_1132_Библиотеки_им_академика_М_И_Перельмана
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Chapter 1
2
Application of Cell-Based
Therapies in Veterinary
Dermatology
CarolinaMesquita, BrunaLopes, PatríciaSousa,
MarianaBranquinho, Ana CatarinaSousa, Ana LúciaLuís,
RuiAlvites and Ana ColetteMaurício
Abstract
S
tem cells have been extensively studied in the field of veterinary medicine due
to their unique characteristics. The last are undifferentiated cells with self-renewal,
anti-inflammatory, and immunomodulatory capacity. Mesenchymal stem cells
(MSCs) are widely used due to its simple isolation and expansion, being collected
from different sources such as adipose tissue, bone marrow, peripheral blood, and
umbilical cord. For that reason, MSCs have been studied and used as innovative
therapies in the treatment of several diseases, such as tendinitis, bone regeneration,
osteoarthritis, neuromuscular diseases, heart diseases, respiratory diseases, kidney
disorders, ophthalmology, oncology, and dermatology. Concerning dermatological
problems, the number of skin diseases in animals has been increasing in recent years.
Skin diseases may be related to genetic conditions, external aggressions, or immunological disorders. Many of these skin pathologies are chronic, reason why the animals
are subjected to long-term therapies, which can have deleterious side effects. This
review aims to highlight the importance of cell-based therapies, using MSCs from
different origins and their secretome, in the field of veterinary dermatology and in
immune-mediated diseases such as atopic dermatitis, furunculosis, anal vasculitis,
and scar tissue regeneration. These approaches should be further explored, as they
have revealed promising results in the search for novel therapies.
Keywords: cell-based therapy, mesenchymal stem cells, skin diseases, veterinary
dermatology, wound healing
. Introduction
In the last years, regenerative medicine has been developing in fields such as
wound healing and skin regeneration. The skin acts as a protective barrier that isolates
the body from harmful agents and injuries. In addition, the skin also contributes to
homeostatic maintenance, regulating the body’s temperature and internal integrity.
Age, tumors development, congenital defects, and degenerative diseases are some
Wound Healing - Recent Advances and Future Opportunities
3
of the factors associated with difficulties in wound healing, reason why regenerative medicine can be very helpful in the achieving better results [1]. The skin can be
frequently injured because of both chronic and acute wounds (burns, diabetic ulcers,
and atopic dermatitis), and these patients experience mental, physical and health
constrains that can lead to a huge socioeconomic burden [2]. Recently, MSCs started
to be used as therapeutic agents capable of regenerating damaged tissues and organs
[3]. For that reason, new cell-based therapies have received attention in both human
and veterinary medicine. MSCs are multipotent cells that derive from the embryonic
layer of the mesoderm. Besides, under the right stimulus, these cells can differentiate
into different lineages, such as osteoblasts, myocytes, chondrocytes, among others
[3–5]. MSCs are undifferentiated cells with specific characteristics such as selfrenewal capacity, originating cells with identical characteristics, and the potential
or ability to differentiate from cells in mature tissues, which gives them the ability
to repair tissues and organs [6, 7]. There are numerous clinical studies demonstrating the therapeutic potential of MSCs in various fields of veterinary medicine
[3]. Moreover, it is known that conventional treatments, based on medical drugs,
are often associated with unwanted side effects due to the re-use of these drugs.
Nevertheless, despite the capacity of MSCs in wound repair and cutaneous regeneration, there are some limitations, such as the heterogenicity in the delivery protocols,
site of delivery, and the lack of information concerning MSCs functional properties
and phenotype [2].
Several dermatological problems have a congenital origin and a chronic/recurring
nature, forcing these animals to receive repeated and prolonged drug treatments with
the consequent development of side effects. Furthermore, skin diseases require a
lot of attention, since they are associated with expensive treatments that are usually
ineffective [1]. Studying the use of cells as a therapeutic agent instead of conventional
drugs, for the control of these patients with dermatological problems, is therefore of
special interest.
This review analyzes the most relevant stem cell types in skin regeneration and
specific dermatological that may benefit from treatment with this new therapeutic
approach.
. The skin and the wound healing process
The skin is the main barrier that protects the body from the external environment,
maintaining the homeostasis and with self-healing capacity. It is a complex organ,
with different layers (epidermis, dermis, and hypodermis), that upon the loss of
integrity, whether due to a disease or a lesion, needs to re-establish its function [1].
The wound healing process is a complex cascade of events that must occur in
sequence and at the adequate time, in order to be successful. It has four overlapping
phases: hemostasis, inflammation, proliferation, and remodeling. After a lesion, the
first reaction of the body is to prevent blood loss, using the platelets to form a blood
clot (hemostasis). Then, the inflammatory phase starts by recruiting inflammatory
cells into the lesion site that will produce growth factors, cytokines and enzymes,
increasing the temperature, redness, swelling, and local pain. If this phase extends in
time, there will be a chronic inflammation that will harm the wound healing. The next
phase is the proliferative, which consists in covering and filling the void space created
by the lesion. For this, wound contraction occurs by local fibroblasts that differentiate into myofibroblasts. In addition, endothelial cells proliferate and migrate to form
Application of Cell-Based Therapies in Veterinary Dermatology
DOI: http://dx.doi.org/10.5772/111553
Figure 1.
4
Wound healing cascade and scar formation.
new blood vessels at the lesion site. It starts in the 4th day post-injury and can last for
2weeks. Then, the fourth phase (remodeling) occurs and extracellular matrix deposits, promoting the re-epithelization and neovascularization, as the collagen fibers
change from type III to type I, helping the tissue to remodel and regain its flexibility
and tensile strength. This phase begins 2–3weeks post-lesion and can last for several
years [1, 8, 9].
The wound healing process can sometimes fail and, although the process is not
fully understood, the prolonged chronic local inflammation is associated with an
abnormal regeneration, as it supports the formation of scars, as demonstrated in
Figure . There are also several factors that increase the risk of inappropriate wound
healing, such as smoking, malnutrition, infections, age, metabolic diseases, medications, and even radiation [1, 8]. Tissue engineering, using stem cell-based therapies
is being explored in various research fields obtaining good outcomes. Stem cells have
gained a lot of attention because of some of their capacities, such as differentiation
and the ability to aid tissue regeneration [10].
. Types of stem cells
Depending on their potential, stem cells can be classified into three types: totipotent (cells have the ability to originate all types of cells from the three germ layers—
endoderm, mesoderm, and ectoderm and extra-embryonic tissues); pluripotent (cells
can originate cells from the three germ layers but not cells from extra-embryonic
tissues), and multipotent (cells can originate cells from several types of tissues but
only from one of the germ layers) [3]. Furthermore, stem cells can be collected from
two major types of tissues, namely embryonic tissues and adult tissues. Their collection using biotechnology is a complex and costly process [11].
Embryonic stem cells (ESC) can be obtained from the inner cell mass of an early
embryo. When removed, these cells can be cultured in vitro and have immortal char-
acteristics. In addition, ESC may be induced to originate various cell/tissue types. For
these reasons, ESCs are studied to better understand the mechanisms of organ formation and healing. Despite this, these cells can promote the formation of teratomas and
can be rejected, when implanted into a patient, and there is some ethics controversy
about the use of embryos in science [12].
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Table 1.
Summary of Adult Stem cells and Embryonic Stem cells and general characteristics.
Adult stem cells (ASC) can be found in almost every tissue, including adipose
tissue, skin, bone marrow, muscle, among others. Some ASC, especially MSCs, can
produce growth factors and can differentiate into many lineages. As opposed to ESC,
ASCs do not lead to the formation of teratomas, unless there has been some damage
prior to its implantation. These different characteristics between ESC and ASCs are
compared in Table . Neonatal stem cells from the amnion, placenta, and umbilical
cord are commonly considered as ASCs [12].
Among all these sources, bone marrow-derived MSCs (BM-MSCs) and adiposederived MSCs (ADSCs) are the most studied and used in veterinary medicine due to
the ease of obtaining, abundance of tissue of origin, and lack of moral restrictions [13].
. Mesenchymal stem cells
MSCs were firstly characterized by Friedenstein’s group as being phenotypically identical to fibroblasts and capable to adhere to plastic surfaces [1]. These
cells are defined by the International Society for Cellular Therapy as cells that
express specific surface markers (CD73, CD90, and CD105), do not exhibit the
hematopoietic markers (CD45, CD34, CD14, CD19, CD11b, CD79a, and others),
and have the ability to adhere to plastic surfaces when in culture and multipotential
ability to differentiate in at least osteoblasts, chondrocytes, and adipocytes under
specific in vitro conditions [14]. MSCs can be isolated from various sources such as
adipose tissue, bone marrow, umbilical cord, dental pulp, olfactory mucosa, and
muscle[15,16].
In addition to their ability to differentiate into various types of tissues, MSCs
can be used to produce secretome, which is composed by a wide variety of secreted
Application of Cell-Based Therapies in Veterinary Dermatology
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bioactive substances such as proteins, cytokines, growth factors, antioxidants,
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proteosomes, and exosomes that interact in an autocrine and paracrine way. MSCs
secretome is an alternative therapeutic option and can help solving some limitations
related to the use of living cells, such as tumorigenicity, immune compatibility, and
infection transmission [10].
Among the numerous performance capabilities of MSCs, it is important to high
light their high capacity to differentiate into various cell types such as osteoblasts,
chondrocytes, adipocytes, hepatocytes, myocardial cells, endothelial, neuronal,
and epithelial cells, helping in the regeneration of damaged tissues. The potential to
secrete cytokines and growth factors can promote angiogenesis and neo-vascularization, thereby increasing tissue blood flow, and the anti-apoptosis characteristics
through the production of cellular factors that promote cellular survival prevent
apoptosis or programmed cell death. In addition, MSCs can migrate to damaged
areas of the body where they can act in tissue repair, which allows its local application
directly in situ or via systemic administration. Their anti-inflammatory action and the
inhibition of pro-inflammatory factors, as well as the immunomodulatory potential,
make these cells good candidates to the treatment of dermatological disorders, as
described in the characteristics in Figure [3, 14, 17, 18].
. Immuno-modulating capacity of MSCs
MSCs act on different types of cells of the immune system by releasing more
than 200 bioregulatory substances with antifibrotic, antiapoptotic, antimicrobial,
chemoattraction, stem cell support, hematopoietic, angiogenesis, mitogenesis and
neuroprotector properties [7]. In addition, MSCs have two fundamental effects on the
immune system, which are an immune-enhancing and anti-inflammatory response
[3]. These cells interact with T cells, B cells, natural killer (NK) cells, dendritic cells
(DCs) macrophages, monocytes, and neutrophils, exerting immunoregulatory action
on the innate and adaptive immune response [17].
Figure 2.
MSCs role in the wound healing process.
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The immunoregulatory potential of MSCs depends on several factors, such as their
tissue of origin, MSC dose, administration time, MSC activation, and their contact
with immune system cells.
. Mechanism of action of MSCs under the innate immune response
The innate immune response is the body’s first line of defense against any external action produced by pathogenic agents such as bacteria, fungi, and virus. It is a
fast-acting and nonspecific response to those pathogens. This defense process causes
tissue inflammation through the activation of immune system cells such as neutrophils, macrophages, monocytes, natural killer and dendritic cells, and the release of
enzymes that form the complement system [19].
MSCs secrete prostaglandin E2 (PGE2), transforming growth factors (TGF-B),
and indolamine2,3-dioxygenase that can modulate NK, inhibiting their proliferation, cytokine release, and cytotoxicity. This mechanism can also be exerted through
cell-to-cell contact. In addition, MSCs also act on monocytes and macrophages.
PGE2, TGF-B, hepatic growth factor, interleukin 6 released by MSCs, reprogram
macrophages with a pro-inflammatory M1 phenotype to an anti-inflammatory M2
phenotype with increased production of interleukin-10, and decreased production of
tumor necrosis factor and gamma interferon [20–22].
The release of these same soluble factors (PGE2, TGF-B, and interleukin 6)
acts on monocytes by inhibiting their differentiation into dendritic cells. Dendritic
cells are antigen-presenting cells, when their maturation is inhibited, the correct
expression of presenting and co-stimulatory molecules does not occur, which
results in a lack of response on the part of T cells [3, 23]. Furthermore, MSCs have
the capacity to inhibit the infiltration of monocytes, macrophages, and neutrophils
into sites of inflammation, dependent on the tumor necrosis factor stimulated gene
6 protein (TSG6). Similarly, MSCs can also enhance the infiltration of the cells into
tumors in a chemokine-dependent manner. In this case, MSCs can promote tumor
progression, metastasis, and treatment resistance. For instance, the stimulation of
chemokine production may stimulate the capacity of MSCs to attract macrophages,
monocytes, and neutrophils. Conversely, an inflammation stage might activate
the expression of indoleamine-2,3-dioxygenase (IDO) produced by MSCs that
can cause immunosuppressive consequences on myeloid cell migration. For these
reasons, it is hard to predict whether the immunomodulatory response of MSCs is
expected to be negative or positive, because of MSCs complex innate immune cell
interactions [23].
. Mechanism of action of MSCs under the adaptive immune response
The adaptive immune response develops a defense mechanism specific for each
pathogen. Therefore, a memory effect is created for each antigen after the first
contact, in order to develop a faster and more effective response the next time the
organism is in contact with the same antigen [3].
The immune system acts through two pathways, the cellular immune response
composed of T lymphocytes that directly attack the pathogens that invade the organism and the humoral immunity response composed of antibodies against pathogenic
antigens produced by B lymphocytes [22].
One of the major mechanisms of action of MSCs is their ability to regulate T cells
through cell-to-cell interaction or secretion of inflammatory components. In this
Application of Cell-Based Therapies in Veterinary Dermatology
DOI: http://dx.doi.org/10.5772/111553
environment, MSCs can change from T-helper 1 (Th1) phenotype (proinflammatory)
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into a T-helper 2 (Th2) phenotype (anti-inflammatory) [3]. There is some contradiction about the effects of MSCs on B cells, although there is clear evidence that MSCs
have close interaction with these cells. Thus, MSCs are capable of inhibiting B cell
proliferation through cell-to-cell contact and with the arrest in the cell cycle. MSCs
can regulate immune responses, but their immunomodulatory capacity is not yet fully
understood.
This anti-inflammatory and immunomodulatory capacity of MSCs is very promis
ing for the treatment and recovery of skin tissue [14].
. Application of cell-based therapies in veterinary dermatology
-
Slow wound healing or persistent wounds are a challenge for clinicians, in both
veterinary and human medicines. These wounds can result in inadequate tissue reorganization, culminating in a long period of incapacity and unsatisfactory outcomes
[24]. These conditions can be related to various pathological conditions, such as autoimmune diseases, diabetes, and venous stasis, for which no definitive therapies are
currently available [25]. Due to this situation, the use of MSCs in veterinary medicine
has been increasing in the recent years in different fields. Regarding their application in dermatology, these cells can be used for skin tissue regeneration and for the
control of dermatological pathologies, in which the immune system intervenes [14].
Due to their capacity for regeneration, differentiation, revascularization, as well as,
their anti-inflammatory and immunomodulatory properties, MSCs have been used as
promoters of regeneration on tissues that suffered some damage [1]. However, there
are still some questions regarding the use of MSCs, such as their immunomodulation
mechanism that is not fully understood. In addition, there are different routes of
administration that can result in different risks for the patient. For instance, systemic
administration can lead to the entrapment of MSCs in the lung or microvasculature
that can cause side effects, such as pulmonary emboli. In addition, almost 90% of the
cells are lost, once administered, because of hypoxia, inflammation, physical stress,
or immunogenic rejection. For this reason, to reach a therapeutic efficacy, a huge
number of cells may be needed, increasing the risk of teratoma formation. Therefore,
new studies are needed to achieve more cost-effective treatments to overcome these
obstacles [3]. Despite these, this review demonstrates the positive effects of using
MSCs therapies and the need to standardize protocols.
There are several skin diseases that can produce chronic and recurring wounds,
such as canine atopic dermatitis (CAD), pemphigus foliaceus (PF), and perianal
fistula. The conventional treatment for the skin diseases consists in glucocorticoids,
cyclosporine, and oclacitinib, which are used due to their immunomodulatory effect.
However, their repeated use has several side effects, such as polydipsia, polyuria,
polyphagia, vomiting, and diarrhea. It can also require increasing doses of medication
over time due to drug habituation [3, 26].
. Application of MSCs in tissue repair and chronic non-healing wounds
A wound is a disruption of the functional integrity and anatomic structure of the
skin, so wound healing is a highly ordered process. The skin tissue repair steps need
to occur in consecutive order and timing, to be successful; otherwise, the healing
process will fail and cause complications, such as chronic non-healing wounds [1].
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There are various types of non-healing wounds, such as bed sores, diabetic foot, and
trophic ulcers of many etiologies. For instance, ulcer treatment requires necrotic
tissue debridement, wound cleansing, amelioration of damaging factors (infection),
improvement of arterial blood circulation, and medical management to aid with the
comorbidities. Nonetheless, chronic wounds take a long time to heal and often recur
after healing with extensive and intensive treatment. Thus, a great possibility in the
treatment of chronic wounds is associated with cell-based therapies [27].
The use of MSCs in wound healing is due to their ability to remove necrotic and
dead cells, improve vascularization and re-epithelization, and diminish scar formation and wound contraction. Transplanted MSCs release several growth factors that
help coordinate different repairing activities. Fibroblasts, endothelial cells, and local
stem cells are triggered to aid in tissue repair, by increasing angiogenesis, restraining
leukocyte transmigration, and stimulating the proliferation, migration, and differentiation of keratinocytes and fibroblasts. MSCs also release immunosuppressive
factors, helping to suppress the proliferation of immune cells, reducing inflammation,
and, consequently, reducing scar formation [1].
Kuperman etal. demonstrated that the local application of mouse oral mucosa
stem cells (mOMSCs) increased the wound healing rate and re-epithelialization and
led to a larger area of granulation tissue in diabetic mice compared to the control
group [28].
Gorecka etal. applied human-induced pluripotent stem cell-derived smooth
muscle cells (hiPSC-SMCs) embedded into 3D collagen scaffolds in diabetic mice,
promoting angiogenesis and accelerating diabetic wound healing [29].
A study in rabbits showed that wounds treated with a combination of plasma rich
in growth factors and adipose-derived mesenchymal stem cells (PRGF+ADSCs) have
higher wound healing and epithelization rates, less inflammation and scar tissue,
greater collagen deposits, and better angiogenesis compared to the control group.
Furthermore, the group treated with the combination PRGF+ADSCs showed a faster
recovery of the damaged tissue [30].
Several studies in rodent models have demonstrated that MSCs applied subcutaneously, topically, or intravenously can improve wound healing [12, 31–33].
In a study focusing on skin wounds in dolphins, the animals were treated with
autologous ADSCs in a blinded clinical study and the group treated with ADSCs
showed improved wound healing [12].
The use of secretome is an alternative treatment when dealing with chronic skin
wounds. Sue etal. used a rat skin excisional wound healing model to demonstrate that
the subcutaneous injection of ADSCs secretome around the wound could accelerate
cutaneous wound healing [34].
Park etal. investigated if ADSCs secretome could accelerate wound healing using
nude mice. In this study, a full-thickness excisional skin wound was created bilaterally on the dorsal surface of the animal. Then, the secretome was used topically in
the wounds and covered with a transparent dressing. The evaluation of the lesions
demonstrated that the treatment using secretome was able to stimulate angiogenesis,
skin thickening, and the recruitment of immune cells, therefore enhancing the wound
healing process [35].
. Application of MSCs in immune-mediated diseases
CAD is a complicated disease that results from environmental factors (allergens)
and a genetic predisposition (filaggrin mutation) that alter the immune response and
Application of Cell-Based Therapies in Veterinary Dermatology
DOI: http://dx.doi.org/10.5772/111553
culminate in a skin barrier dysfunction [36]. It is a common multifactorial inflamma-
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tory and pruritic skin disease in dogs, associated with the production of IgE antibodies
[37]. Its prevalence is around 10–15%, and the management of these patients is a real
challenge for tutors and veterinarians [38]. During the acute phase of CAD, there is an
activation of Th2 cells caused by the immune dysregulation, which culminates in the
production of numerous pro-inflammatory cytokines. Over time, the chronicity of the
pathology is maintained by a broader roster of T helper variations. Pharmacological
treatments consist in corticosteroids (less specific) and cyclosporine A (more
specific), and some new approaches used to achieve the most target agents, such as
lokivetmab and oclacitinib. Despite the newest therapies, approximately 25–40% of
dogs with CAD endure clinical signs and do not reach a full resolution of the pathology [39]. For that reason, the use of MSCs would be a good alternative therapy, since
their use in an inflammatory environment, can alter the cytokine profile of T cells and
dendritic cells, which can lead to an anti-inflammatory environment [12].
In 2018, a study gathered 26 animals diagnosed with CAD refractory to conven
tional treatments to which an intravenous dose of 1.5×10
6
ADSCs/kg bodyweight
-
was administered. Pruritus was evaluated using the Canine Atopic Dermatitis Extent
and Severity Index, version 4 (CADESI-4), and a decrease in pruritus within a time
span of 1week to 1month was observed, with the animals being controlled for a
period of 6months. Owners reported improvement of the animals with a satisfactory
global assessment of the treatment without the occurrence of adverse events [40].
In 2019, a group of 12 canine patients diagnosed with CAD were intramuscularly
inoculated with 0.5×10
6
of cryopreserved ADSCs. Injections were repeated weekly
for 6weeks. During this period, the effectiveness of the treatment was evaluated by
the pruritus index and by the CAD Lesion Index (CADLI) test, and a notable reduction in both was observed. The animals were monitored at all times, and no systemic
side effects or changes at the injection site were observed [41].
In 2020, a group of 16 animals diagnosed with CAD was evaluated with CADESI-4
and divided into three groups, namely mild, moderate, and severe according to the
severity of their injuries. For 82days, 2x106 MSCs were inoculated intravenously
every 21days to all animals. At the end of the 82days, skin biopsy histopathology
analysis was performed, observing a significant reduction in epidermal thickness in
the moderate and severe groups. The results demonstrate that MSCs attenuated the
clinical signs of CAD resulting in a safe therapy and causing no adverse effects [42].
In 2021, a double-blind study divided patients with CAD into three groups, a
control group that received PBS solution, one group that received low-dose ADSCs
(5×105 cells/kg), and the third group received a higher dose (5×106 cells/kg) of
MSCs. Three subcutaneous treatments were performed at 4-week intervals. Pruritus
was assessed by tutors using the pruritus visual analog scales (PVAS) and by veterinarians using CADESI-4. Both observed a decrease in pruritus during the 30days
following injections in the group receiving higher dose of MSCs. The animals were
monitored throughout the study and did not manifest adverse side effects [43].
Recently, in 2022, a study evaluated the immunomodulatory effect of cADSCs
and extracellular vesicles derived from cADSCs (cADSC-EVs) demonstrating that
these cells have a beneficial effect in atopic animals. The cASCs and cADSC-EVs
affect the expression levels of epidermal differentiation proteins, such as keratin1,
filaggrin, loricrin, and involucrin promoting the recovery of the deficient skin
barrier in these atopic animals. With the recovery of the skin barrier, it was possible
to reduce the loss of water and prevent the entry of allergens via transepidermal
route. cADSCs and cASC-EVs regulate the immune and inflammatory response
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